Phytosanitary composition comprising essential oils that potentiate antifungal activity

ABSTRACT

The present invention relates to phytosanitary compositions with fungicidal properties that comprise a mixture of essential oils obtained from plants and agents with known fungicidal properties, such as alkali metal or ammonium bicarbonates, and compounds based on copper or the salts thereof, for use, principally, in contact-protection against fungal infections in cultivated plants and post-harvest, and also in other antifungal applications. In said compositions, the effect of the agents that have known fungicidal properties is potentiated synergistically by the aforementioned essential oils. The present invention also relates to the use of said essential oils as potentiators for agents with known fungicidal properties.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. patent applicationSer. No. 15/890,484 filed Feb. 7, 2018, which is a divisionalapplication of U.S. patent application Ser. No. 13/982,181 filed Jul.26, 2013, now abandoned, which is a U.S. National Stage Application ofPCT/ES2012/070005 filed Jan. 5, 2012, which claims benefit to SpanishPatent Application No. 201130390 filed Mar. 18, 2011, the contents ofthese applications being incorporated herein by reference in theirentirety.

DESCRIPTION

This invention relates to phytosanitary compositions having fungicidalproperties comprising a mixture of essential oils obtained from plantsand agents having known fungicidal properties such as alkali metal orammonium bicarbonates, or compounds based on copper or copper salts, foruse mainly in contact protection against fungicidal infections incultivated plants and post-harvesting, and in other antifungalapplications. In these compositions the effect of the agents havingknown fungicidal properties is synergistically potentiated by theessential oils mentioned. This invention also relates to use of theseessential oils as potentiators for agents having known fungicidalproperties.

Essential oils are complex mixtures of natural molecules which arefundamentally obtained from plants. They are secondary metabolites whichcan normally be obtained by extraction with organic solvents andsubsequent concentration, or by physical treatments with steam followedby separation of the water-insoluble phase. Generally they are volatileliquids soluble in organic solvents and have a density lower than thatof water.

In nature they can be synthesised in different plant organs such asseeds, leaves, flowers, epidermal cells and fruits, among others, andthey play an important part in protecting plants against bacterial,viral or fungal infections.

The fungicidal and bactericidal action of many plant essential oils isknown, have arrived in some case to be marketed commercially. Amongthese are jojoba oil (Simmondsia californica), rosemary oil (Rosemarinusofficianalis), thyme oil (T. vulgaris), the clarified hydrophobicextract of neem oil (A. indica), cottonseed oil (Gossypium hirsutum)with garlic extract (Dayan, F. E. et al. “Natural products in cropprotection”. Bioorg. and Med. Chem. 17 (2009), 4022-4034).

The chemical composition of essential oils differs not only in thequantity but also in the quality and the stereochemical type of themolecules in the extracted substances. The extraction product may varyaccording to climate, the composition of the soil, the organ of theplant used for extraction, and the age and stage of growth of the plant.It also depends on the extraction process used.

Furthermore, the use of inorganic salts such as the bicarbonates ofalkali metals, mainly lithium, sodium or potassium, and ammoniumbicarbonate as fungicidal agents is also known from the prior art (U.S.Pat. No. 5,346,704). The use of these inorganic salts, in particularthose containing the bicarbonate anion, does not give rise to any risksto human health or to the environment.

The fungicidal nature of products based on copper or its salts are alsoknown, and these have been extensively used in agriculture. In the year1761 it was discovered that these solutions inhibit the growth of fungion seed. Since then copper-based fungicides have been used in well-knownformulae such as Bordeaux mixture (Copper as a Biocidal Tool. GadiBorkow and Jeffrey Gavia. Current Medicinal Chemistry, volume 12:2163-2175).

Because of their natural origin plant essential oils are very attractivefor application in agriculture in order to obtain healthy and harmlessproducts, as this is a requirement which has been made increasinglystrictly, by both consumers and regulatory authorities.

There is therefore a need to find new phytosanitary compositions havingantifungal properties to protect crops, including duringpost-harvesting, which have a minimum of secondary toxic effects andwhich are harmless to human beings and the environment.

The present authors have surprisingly found that some essential oilsobtained from plants when mixed with other products having knownantifungal properties potentiate the antifungal properties of thesecompounds, such as inorganic salts, for example alkali metal or ammoniumbicarbonates, and antifungal products based on copper or its salts.

Thus one object of this invention is to provide a phytosanitarycomposition having antifungal properties comprising: 1) one or moreessential oils obtained from plants and 2) one or more agents havingknown fungicidal properties. This composition synergistically improvesthe antifungal properties of the agents having known antifungalactivity, has a minimum of secondary toxic effects and is harmless tohuman beings and the environment.

The composition according to this invention may be applied inagriculture to protect crops from the stage of germination toharvesting, and during the storage and transport of these crops, seeds,flowers or grains. Likewise, another possible application is in theelimination of fungi which attack painted surfaces and to protectcarpets and fabrics in the home and in any other application againstfungal attack through contact.

Among the essential oils which may be used in the phytosanitarycomposition according to this invention are thyme oil (Thymus vulgaris),oregano oil (Origanum vulgaris), clove oil (Syzygium aromaticum), nutmegoil (Myristica fragrans), cinnamon oil (Cinnamomum zeylanicum), laureloil (Laurus nobilis), orange oil (Citrus x sinensis), mint oil (Mentha xpiperita), valerian oil (Valeriana officinalis), citronella oil(Cymbopogon nardos), lavender oil (Lavanda angustifolia), jojoba oil(Simmondsia californica), rosemary oil (Rosemarinus officianalis), neemoil (Azadirachta indica), cottonseed oil (Gossypium hirsutum) ormixtures thereof.

Without being bound to any theory in particular, it is possible that theproperty of the essential oils obtained from plants in potentiatingantifungal activity is due to some of the compounds having knownactivity present in these essential oils. Thus in one embodiment of thisinvention the phytosanitary composition may comprise a mixture of activecompounds isolated from the essential oils according to this invention,such as phenolic monoterpenoids such as carvacrol and thymol,allylbenzenes such as eugenol, monosubstituted phenols such astrans-cinamaldehyde, cyclic monoterpenes such as limonene, bicyclicmonoterpenes such as camphene and linear terpenes such as nerol, any oftheir families and mixtures thereof, and agents having known fungicidalproperties. The mechanism of action of the essential oils is a multipleone due to the complex mixture of different active ingredients whichthey contain. However the nature of the action of the major componentsin some of these oils has been described. The best described in theliterature is the nature of the action of carvacrol on the growth ofbacterial and yeast cells (The phenolic hydroxyl group of carvacrol isessential for action against the food-borne pathogen Bacillus cereus. A.Ultee et al., Applied and Environmental Microbiology, April 2002,1561-1568). According to these studies carvacrol is capable of crossingthe cell membrane when it is protonated (in acid medium) and on reachingthe cytoplasm releases a proton, resulting in acidification of the cell.This manner of action does not rule out other possible modes of actionsuch as increase in the permeability of the membrane or specificinhibiting effects on catalytic processes.

Among the agents having known fungicidal properties which may be used inthe composition according to the invention there are the carbonates orbicarbonates of alkali metals, preferably of lithium, sodium orpotassium, ammonium carbonate or bicarbonate and fungicidal agents basedon copper or its salts or mixtures thereof. More preferably the agenthaving known fungicidal properties is potassium bicarbonate.

The quantity of essential oils present in the composition according tothis invention lies within the range from 0.01% to 99.99% by weight ofthe total composition. Also the quantity of the agent having knownfungicidal properties in the composition according to this invention mayvary between 99.99% and 0.01% by weight of the total composition.

The composition according to this invention may be prepared by mixingthe essential oil or oils and the agent having fungicidal propertiesthrough any method of mixing known in the art. In general essential oilsare liquid at ambient temperature, as a result of which the compositionwill generally be in the form of a liquid. However, the composition mayalso be in liquid or solid form, such as a suspension, dispersion,emulsion, spray, microencapsulate or any other type of mixture whichremains stable over time or may be incorporated in polymers, waxes orany other similar supports.

Furthermore the phytosanitary composition according to this inventionmay be used as such, or may be used to formulate a phytosanitary producttogether with different additives used in the art which offer differentproperties, such as surfactants, polymers, alkanising agents, pH-controlagents, among many other additives used in the formulation of productsused in the agricultural industry.

In embodiments, the phytosanitary composition may include carvacrol at aconcentration between 0.1 ppm and 530 ppm and potassium carbonate at aconcentration between 3.5 mM and 25 mM. For example, the carvacrol maybe present in a concentration between 22 ppm and 310 ppm. The potassiumcarbonate may be present in a concentration between 10 mM and 25 mM, forexample.

The phytosanitary composition according to this invention falls withinthe group of contact phytosanitary agents, that is the form of theprotection against fungal diseases is through contact, given that thecomposition remains on the surface of different parts of the plant,protecting it externally against the external attack of fungi.

Being a liquid, a powder or a microencapsulate, the phytosanitarycomposition according to this invention can be applied by any method ofapplication known in the art, such as spraying.

The fungicidal composition according to this invention may furthercomprise a fertiliser, which may be selected from the group comprisingcompounds containing nitrogen and/or phosphorus such as urea, melamine,hexamine, dicyanodiamide, ameline, cyanuric acid, melamine nitrate,triethyl phosphite and the like or mixtures thereof.

The composition according to this invention may also comprise anycompound or product having chemical and/or biological activity used inagriculture, such as herbicides, insecticides, plant growth regulatorsand the like, or mixtures thereof.

This invention is described below in greater detail with reference tovarious examples. However, these examples are not intended to restrictthe technical scope of this invention.

EXAMPLES Example 1 (Comparative). Inhibition of Growth of the FungusBotrytis cinerea by KHCO₃ Alone

The fungus Botrytis cinerea was cultured in PDB (potato dextrose broth)medium with different concentrations of KHCO₃ and the % inhibitionrepresenting the extent to which growth was impeded in comparison with acontrol which did not have the compound under test, in this case KHCO₃,was determined. The % inhibition was calculated in the following way:% inhibition=[(OD_(control)−OD_(x))/OD_(control)]×100where OD_(control) is the optical density of the control culture(without test compound) and OD_(x) is the optical density of the culturewith the test substance. The optical density of the liquid culture wasmeasured 24 hours after the start of culturing.

The following results were obtained (Table I):

TABLE I Inhibition of the growth of B. cinerea by KHCO₃ KHCO₃ 0 10 15 2025 30 concentration (mM) Inhibition 0 34.2 ± 37.0 ± 38.8 ± 39.7 ± 0.746.9 ± 2.7 (%) ± SD 6.7 11.3 6.8

As will be seen from the table above, with a KHCO₃ concentration between10 and 25 mM similar results were obtained as regards inhibition of theB. Cinerea culture. However greater inhibition was obtained at 30 mM.

Example 2. (Comparative). Inhibition of Growth of the Fungus Botrytiscinerea by Carvacrol Alone

The fungus B. cinerea was cultured in a similar way to Example 1 withthe difference that different concentrations of carvacrol, the compoundisolated from the essential oil of oregano, were used in the medium. The24 hour optical density of the culture was measured and the results areshown in Table II.

TABLE II Inhibition of the growth of B. cinerea by Carvacrol Carvacrol0.1 0.31 1 3.1 10 31 100 concentration (ppm) Inhibition 0 10.5 ± 7.713.7 ± 4.1 22.4 ± 3.4 21.3 ± 5.0 51.4 ± 5.5 74.4 ± 1.1 (%) ± SD

Example 3. Inhibition of Growth of the Fungus Botrytis cinerea by theComposition According to this Invention (Carvacrol+KHCO₃)

The fungus B. cinerea was cultured in a similar way to Example 1 withthe difference that different concentrations of carvacrol were used inthe medium and that a constant concentration of KHCO₃ (30 mM) was usedfor all the cultures. The optical density of the culture was measured at24 hour and the results are shown in Table III.

TABLE III Inhibition of the growth of B. cinerea by the compositionaccording to this invention (Carvacrol + KHCO₃) KHCO₃ 30 30 30 30 30 3030 concentration (mM) Carvacrol 0.1 0.31 1 3.1 10 31 100 concentration(ppm) Inhibition 86.1 ± 2.2 84.2 ± 3.6 48.9 ± 3.0 52.7 ± 2.6 82.5 ± 3.786.5 ± 1.3 91.1 ± 1.1 (%) ± SD

As will be seen, a carvacrol concentration as low as 0.1 ppm, whoseinhibiting effect alone is zero (see Example 2) effectively doubles theinhibiting capacity of the KHCO₃, achieving levels of inhibition whichare not even obtained with KHCO₃ concentrations that are toxic toplants.

Example 4. Inhibiting Effect of Copper Oxychloride Alone on the FungusAlternaria alternata

Alternaria alternata was cultured in a similar way to Example 1 with thedifference that different concentrations of copper oxychloride, acopper-based fungicide extensively used in agriculture, were used in themedium. The 24 hour optical density of the culture was measured and theresults are shown in Table IV.

TABLE IV Inhibition of A. alternata by copper oxychloride Copper 0.1 0.51 5 10 15 20 oxychloride concentration (ppm) Inhibition 3.1 ± 6.4 0.0 ±6.7 9.9 ± 0.9 10.0 ± 8.4 23.1 ± 4.7 37.4 ± 3.6 61.3 ± 6.7 (%) ± SD

Example 5. Inhibition of the Fungus Alternaria alternata by CarvacrolAlone

Alternaria alternata was cultured in a similar way to Example 2. The 24hour optical density of the culture was measured and the results areshown in Table V.

TABLE V Inhibition of A. alternata by Carvacrol. Carvacrol 10 31 100 3101000 concen- tration (ppm) Inhibition 17.7 ± 11 27.2 ± 14 74.6 ± 8 97.2± 7 93.0 ± 6.0 (% ± SD)

Example 6. Inhibition of the Fungus Alternaria alternata by theComposition According to this Invention (Carvacrol+Copper Oxychloride)

The fungus A. alternata was cultured in a similar way to Example 4 withthe difference that different concentrations of carvacrol were used inthe medium and that a constant concentration of copper oxychloride (5ppm) was used throughout. The 24 hour optical density of the culture wasmeasured and the results are shown in Table VI.

TABLE VI Inhibition of A. alternata by the composition according to thisinvention (Carvacrol + copper oxychloride) Copper 5 5 5 5 5 oxychlorideconcen- tration (ppm) Carvacrol 1 3.1 10 31 100 concen- tration (ppm)Inhibition 1.4 ± 13 26.0 ± 12 34.7 ± 14 53.2 ± 12 85.7 ± 3.6 (%) ± SD

As will be seen, a carvacrol concentration of 35 ppm and 5 ppm of copperoxychloride inhibit the growth of A. alternata by more than 50%, whilecarvacrol alone in that concentration provides 27% inhibition and copperoxychloride only 10%.

Example 7. Inhibition of the Fungus Penicillium digitatum by KHCO₃ Alone

The fungus Penicillium digitatum was cultured in a similar way toExample 1. The 24 hour optical density of the culture was measured andthe results are shown in Table VII.

TABLE VII Inhibition of P. digitatum by KHCO₃. KHCO₃ 10 20 30 40 50concen- tration (mM) Inhibition 19.4 ± 2.9 19.1 ± 10 19.6 ± 8.9 19.8 ±2.2 21.9 ± 6.6 (% ± SD)

As will be seen, the same degree of inhibition is obtained for differentKHCO₃ concentrations.

Example 8. Inhibition of the Fungus Penicillium digitatum by ThymolAlone

The fungus P. digitatum was cultured in a similar way to Example 1 withthe difference that different concentrations of thymol, a compoundisolated from thyme oil, were used in the medium. The 24 hour opticaldensity of the culture was measured and the results are shown in TableVIII.

TABLE VIII Inhibition of P. digitatum by Thymol alone Thymol 0.31 1 3.110 31 100 310 concentration (ppm) Inhibition (% ± 28.2 ± 3.9 24.2 ± 6.036.3 ± 2.3 36.2 ± 2.0 50.7 ± 2.0 78.3 ± 2.2 95.6 ± 0.5 SD)

Example 9. Inhibition of the Fungus Penicillium digitatum by theComposition According to this Invention (KHCO₃+Thymol)

The fungus P. digitatum was cultured in a similar way to Example 7 withthe difference that different concentrations of thymol were used in themedium and that a constant concentration of KHCO₃ (30 mM) was usedthroughout. The 24 hour optical density of the culture was measured andthe results are shown in Table IX.

TABLE IX Inhibition of P. digitatum by the composition according to thisinvention (KHCO₃ + Thymol) KHCO₃ concentration 30 30 30 30 30 30 (mM)Thymol 0.31 1 3.1 10 31 100 concentration (ppm) Inhibition 58.0 ± 2.162.9 ± 8.4 47.1 ± 3.7 56.4 ± 3.7 75.5 ± 1.9 92.6 ± 1.6 (%) ± SD

It will be seen how the results are improved by adding thymol to KHCO₃.With 31 ppm of thymol only 50% inhibition is achieved, and with 30 mM ofKHCO₃ 20% inhibition is achieved. However when the two compounds arecombined inhibition of growth of the fungus P. digitatum is increased upto some 75%.

The invention claimed is:
 1. A phytosanitary composition havingantifungal activity comprising: (1) an oregano (Origanum vulgare)essential oil obtained from plants comprising its active compoundcarvacrol which is present at a concentration between 0.1 and 530 ppm inthe phytosanitary composition; and (2) potassium carbonate at aconcentration between 3.5 and 25 mM.
 2. A phytosanitary compositionaccording to claim 1, characterised in that the carvacrol is at aconcentration between 22 and 310 ppm.
 3. A phytosanitary compositionaccording to claim 1, characterised in that the potassium carbonate isat a concentration between 10 and 25 mM.
 4. A phytosanitary compositionaccording to claim 1, characterised in that the composition is in liquidor solid form which remains stable over time or is incorporated in asupport.
 5. A phytosanitary composition according to claim 1,characterised in that the phytosanitary composition is formulatedtogether with additives.
 6. A phytosanitary composition according toclaim 1, which further comprises a fertiliser selected from the groupconsisting of compounds containing nitrogen and/or phosphorus and amixture thereof.
 7. A phytosanitary composition according to claim 1,which further comprises a compound or product having chemical and/orbiological activity used in agriculture.